Preparation of the 16S rRNA gene amplicon library for MiSeq sequencing

Ileal contents (200 mg) from each bird were collected for DNA isolation utilizing QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA). The concentration of extracted DNA was diluted to 10 ng μL⁻¹ for the preparation of a sequencing library targeting the V4 region of the 16S rRNA gene (37). Isolated DNA samples were amplified via a PCR using dual-index primers and normalized the amplicons with a SequalPrep™ Normalization kit (Life Technology, Carlsbad, CA) according to the manufacturers' recommendation. The library was constructed by combining 5 μL of each normalized aliquot sample for further assessment. Library concentration and product size were confirmed using a KAPA Library Quantification Kit (Kapa Biosystems, Woburn, MA) via quantitative PCR (qPCR, Eppendorf, Westbury, NY) and an Agilent 2100 Bioanalyzer system (Agilent, Santa Clara, CA), respectively. The 20 nM of pooled library aliquot and the 20 nM of PhiX control v3 were combined with 0.2 N fresh NaOH and HT1 buffer and mixed a second time with 5% of the PhiX control v3. The 600 μL of the mixture containing pooled library, PhiX control v3, NaOH and HT1 buffer was subsequently loaded onto a MiSeq v2 reagent cartridge to run sequencing.